The influenza M2 protein forms a pH-activated proton channel that is essential for the virus lifecycle. Inhibition of the H+ channel activity by the amantadine class of antiviral drugs has been made ineffective by mutations in the M2 transmembrane domain. High-resolution structure determination of M2 is thus paramount for developing new antiviral drugs to target amantadine-resistant M2 mutants. The small M2 protein contains all the machinery necessary for pH activation, H+ selectivity, and gating, and thus also provides an excellent model system for understanding larger and more complex voltage-gated H+ channels and other pH-gated ion channels. Work funded by this research proposal has already 1) led to the elucidation of the pharmacologically relevant drug binding site in M2 and the drug-complexed high-resolution structure in the lipid bilayer, and 2) revealed novel pH-dependent dynamics of the proton-selective residue, His37. However, new alternative H+ conduction models have been proposed in the meantime, and the structure basis for channel gating by Trp41 has not been studied. The first aim of this proposal is to elucidate the H+ conduction mechanism of M2 by examining His37 structure at mildly acidic pH when the channel is first activated. Sidechain H-bonding, protonation/deprotonation dynamics, and the effects of inhibitors on His37 structure will be measured. Both amantadine and Cu2+ will be used as inhibitors, and Cu2+ paramagnetic relaxation enhancement effects will be explored for structure determination. The second aim is to elucidate Trp41 structure and interaction with His37 as a function of pH, to understand how these two residues act in unison to achieve channel gating, again in a bilayer environment. In addition to the H+ channel activity, M2 also mediates virus budding by causing membrane curvature in a cholesterol-dependent fashion. We will investigate M2-membrane and M2-cholesterol interactions by distance and relaxation NMR measurements. The hypothesis that M2 preferentially localizes to highly curved regions of the membrane will be tested. Finally, M2 interacts with matrix protein M1 through its cytoplasmic tail during virus assembly and budding. No structural information is available so far for the cytoplasmic domain. We will determine the three-dimensional structure of full-length M2 in lipid bilayers using multidimensional magic-angle-spinning solid-state NMR techniques, to lay the ground for future investigations of the M2-M1 interactions important for the influenza life cycle.